SUMMARY Overnutrition and physical inactivity promote the accumulation of sphingolipids such as ceramides which block insulin signaling and anabolic metabolism. Implementation of pharmacological or genetic interventions to reduce sphingolipid levels in rodents prevents or reverses an impressive array of metabolic pathologies (e.g. insulin resistance, diabetes, steatohepatitis, hypertension, cardiomyopathy, and atherosclerosis). To elucidate the tissue-specific mechanisms through which ceramides contribute to these diseases, we have produced mice allowing for the conditional, cell-type restricted ablation of enzymes required for ceramide biosynthesis or degradation (i.e. serine palmitoyltransferase and dihydroceramide desaturases-1) or degradation (i.e. acid ceramidase). We will apply these tools to dissect the regulatory mechanisms controlling ceramide synthesis and action in skeletal muscle. Aims of the project include the following: ? To use these novel mouse models to evaluate the effect of muscle-specific ceramide depletion or induction on insulin sensitivity, muscle growth, and genomic/proteomic signatures under conditions of overnutrition and inactivity. ? To apply a ceramide flux assay in isolated human myotubes to identify the regulatory mechanisms that influence rates of ceramide biosynthesis; and, ? To determine the efficacy of a new class of inhibitors of dihydroceramide desaturases-1, our preferred target in the ceramide synthesis pathway, as therapeutics that improve muscle insulin sensitivity and prevent muscle loss in rodents. Findings obtained from these studies could uncover new nutrient-sensing machinery that modulates insulin sensitivity and muscle growth. Moreover, the translational component could lead to new pharmacological approaches for improving muscle health.